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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Definitions

• Salmonellae are among the most serious pathogens transmitted to humans through foodstuffs. Since the detection and identification of Salmonellae by conventional microbiological detection processes is very time-consuming—at least five days are required for the increase in quantity and subsequent serotyping required by legal regulations (LMBG, FDA)—there is a great need for alternative rapid methods.
• the mentioned nucleic-acid-based processes are so sensitive that, unlike conventional microbiological processes, a lengthy increase in quantity of the microorganism to be detected from the sample to be investigated is unnecessary. An investigation of the presence or absence of, for example, Salmonellae is therefore generally concluded within one working day when using the mentioned nucleic-acid-based processes.
• nucleic acid sequences for detecting Salmonellae by polymerase chain reaction are known.
• a disadvantage is, however, that when using those nucleic acid sequences as primers in the polymerase chain reaction false positive results [WO 95/33854] or false negative results [WO 92/01056; WO 95/00664; WO 92/01056; WO 93/04202] occur.
• WO 92/08805; WO 94/25597; DE 4337295 so that thus far it is unclear whether the nucleic acid sequences in question are suitable for detecting all Salmonella strains.
• An advantage of, for example, the primers and probes described in International Patent Application WO 95/00664 is that they allow the highly selective detection of bacteria of the Salmonella genus without the occurrence of false positive results.
• a disadvantage when using the oligonucleotides according to WO 95/00664 in amplification processes such as polymerase chain reaction is, however, the fact that none of the described primer pairs enable detection of all the representatives of the 7 Salmonella subspecies. For example, when using the primers ST11/ST15, a number of representatives of subspecies IIIa (subsp. arizonae ) are not detected, and when using the primers ST11/ST14 a number of representatives of subspecies I (subsp. enterica Serovar. Blockley) and of subspecies IIIa (subsp. arizonae ) are not detected.
• An aim of the invention described herein was to optimise the detection processes described in WO 95/00664 by finding nucleic acid sequences the use of which as primers and/or probes ensures as complete detection as possible of all the representatives of the Salmonella genus.
• the problem underlying the invention is solved by a set of nucleic acid molecules by means of which, in a process for the detection of representatives of Salmonella enterica subsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori and indica , all the representatives of those subspecies can be detected, the set being obtainable by
• nucleic acid molecule 1 a nucleic acid molecule 1 in a manner known per se using a nucleic acid isolate of a representative of one of the mentioned Salmonella enterica subspecies, which first nucleic acid molecule is specifically suitable as primer or probe for the detection of that representative or of further or all representatives of that one Salmonella enterica subspecies and possibly also of representatives of further Salmonella enterica subspecies,
• nucleic acid molecule 2 a second nucleic acid molecule (nucleic acid molecule 2) in a manner known per se using a nucleic acid isolate of a different representative of one of the mentioned Salmonella enterica subspecies, which second nucleic acid molecule is specifically suitable as primer or probe for the detection of that representative or of further or all representatives of that different Salmonella enterica subspecies and possibly also of representatives of others of the mentioned Salmonella enterica subspecies, and
• a derived nucleic acid molecule may be a nucleic acid molecule that can be hybridised with the obtained nucleic acid molecule and that preferably has the same number of bases, possible hybridisation conditions being:
• a derived nucleic acid molecule may be, for example, a nucleic acid molecule the sequence of which has been determined by computer design and that has subsequently been manufactured and obtained by chemical synthesis.
• nucleic acid molecule(s) Y Z, . . .
• that(those) nucleic acid molecule(s) being characterised in that the use of that(those) nucleic acid molecule(s)—in addition to the use of a nucleic acid molecule (X)—in a process for the detection of bacteria of the Salmonella genus enables the detection also of Salmonella strains or Salmonella isolates that cannot be detected or can be detected only with relatively low sensitivity using the nucleic acid molecule (X).
• the set of nucleic acid molecules according to the invention can be characterised in that the nucleic acid isolates comprise or are phylogenetically conserved base sequences or regions of those base sequences.
• phylogenetically conserved base sequence see, for example, WO 95/00664 or Herder's Lexikon der Biochemie und Molekularbiologie, supplemented 1995, page 132, spectrum, production, etc.
• the set of nucleic acid molecules according to the invention or a set of nucleic acid molecules according to the invention by means of which, in a process for the detection of representatives of Salmonella enterica subsp. enterica, salamae, arizonae, diarizonae, houtenae, bongori and indica , all the representatives of those subspecies can be detected, can be characterised in that the set for an individual nucleic acid molecule, for a number of its individual nucleic acid molecules or for each of its individual nucleic acid molecules in each case comprises at least one further nucleic acid molecule that, in a region of at least 10 successive nucleotides of their nucleotide chain, corresponds to less than 100% but to at least 80% of the base sequence.
• Such a set of nucleic acid molecules according to the invention can be characterised in that the set for an individual nucleic acid molecule, for a number of its individual nucleic acid molecules or for each of its individual nucleic acid molecules in each case comprises at least one further nucleic acid molecule that, in a region of at least 10 successive nucleotides of their nucleotide chain, differs from the other or further nucleic acid molecule in precisely one base position.
• a set of nucleic acid molecules according to the invention can be characterised in that it comprises one or more, but not exclusively, nucleic acid molecules that are fragments of the SEQ ID NO 1 according to WO 95/00664 or of its complementary sequence.
• a set of nucleic acid molecules according to the invention can also be characterised in that the individual nucleic acid molecules hybridise to the same strand of nucleic acid isolates of representatives of Salmonella enterica subspecies that are being subjected to the process for their detection.
• nucleic acid molecule that belongs to a set of nucleic acid molecules according to the invention or that can be used for such a set, the nucleic acid molecule being characterised in that, in a region of at least 10 successive nucleotides of its nucleotide chain, the sequence of the nucleic acid molecule corresponds exactly to a sequence region of at least one representative of the mentioned Salmonella enterica subspecies, the sequence region comprising or being a phylogenetically conserved base sequence or a region of that base sequence.
• nucleic acid molecule can be characterised in that, in a region of at least 10 successive nucleotides of its nucleotide chain, it is 100% or at least 80% identical to a corresponding number of successive nucleotides of one or more of the following sequences or their complementary sequences:
• nucleic acid molecule characterised in that, in respect of its sequence, it is homologous to an above-characterised nucleic acid molecule according to the invention and, in at least 10 successive nucleotides of its nucleotide chain,
• (iii) differs from an above-characterised nucleic acid molecule according to the invention in not more than two nucleotides.
• a nucleic acid molecule according to the invention can be characterised in that it is from 10 to 250 nucleotides long and preferably from 15 to 30 nucleotides long.
• a nucleic acid molecule according to the invention can also be characterised in that it is single-stranded or has a complementary strand.
• a nucleic acid molecule according to the invention can also be characterised in that it is a modified or labelled nucleic acid molecule in which up to 20% of the nucleotides of at least 10 successive nucleotides of its nucleotide chain are building blocks known per se as probes and/or primers, especially nucleotides that do not occur naturally in bacteria.
• a nucleic acid molecule according to the invention can also be characterised in that it is a modified or labelled or additionally modified or labelled nucleic acid molecule that comprises, in a manner known per se for analytical detection processes, one or more radioactive groups, coloured groups, fluorescent groups, groups for immobilisation on a solid phase, groups for an indirect or direct reaction, especially for an enzymatic reaction, preferably using antibodies, antigens, enzymes and/or substances having an affinity for enzymes or enzyme complexes, and/or other modifying or modified groups of nucleic-acid-like structure that are known per se.
• kit for analytical detection processes especially for the detection of bacteria of the Salmonella genus, that kit being characterised by
• a kit according to the invention can thus comprise a set of nucleic acid molecules according to the invention or one or more nucleic acid molecules according to the invention, there additionally being provided the other customary components for nucleic acid hybridisations or nucleic acid amplifications, for example a polymerase, a reverse transcriptase, a ligase or an RNA-polymerase, see, for example, WO 95/00664.
• a set of nucleic acid molecules of the kit according to the invention will preferably be produced synthetically in at least two separate synthesis batches.
• the kit according to the invention preferably does not comprise any degenerate nucleic acid molecules.
• the problem underlying the invention is solved by the use of a set of nucleic acid molecules according to the invention or of a kit according to the invention to detect the presence or absence of bacteria belonging to a group of bacteria of the Salmonella genus, especially of representatives of the above-mentioned Salmonella enterica sub-species.
• nucleic acid hybridisation and/or nucleic acid amplification can be carried out.
• PCR polymerase chain reaction
• differences between the genomic DNA and/or RNA of the bacteria to be detected and of the bacteria that are not to be detected can be determined at at least one nucleotide position in the region of a nucleic acid molecule according to the invention and representatives of a group of bacteria of the Salmonella genus can be detected, especially representatives of the mentioned Salmonella enterica subspecies.
• Salmonella-specific oligonucleotides are nucleic acid molecules, from 10 to 250 bases (preferably from 15 to 30 bases) long, the base sequence of which is characteristic for Salmonellae: when using such oligonucleotides as primers or probes—with suitable reaction conditions—hybridisation/amplification takes place only when DNA of the Salmonellae to be detected is present in the test sample, but not when DNA of other bacteria is present.
• oligonucleotides may also be used as primers or probes. Such oligonucleotides enable hybridisation and/or amplification not only when Salmonella-DNA is present in the sample but also in the presence of DNA of a bacterium or of a number of bacteria not belonging to the Salmonella genus.
• nucleic acids preferably genomic DNA
• the direct detection of Salmonella nucleic acids in the sample to be investigated can then be effected using the Salmonella-specific oligonucleotides according to the invention as probe.
• Various processes known to the person skilled in the art are suitable for that purpose, such as, for example, “Southern blot” or “dot blot”.
• the DNA/RNA sequences sought are firstly amplified by means of the above-mentioned processes for amplifying nucleic acids, preferably PCR.
• the amplification of DNA/RNA is effected by using Salmonella-specific oligonucleotides. In that process specific amplification products are formed only when Salmonella-DNA/RNA is present in the sample to be investigated.
• the specificity of the detection process can be increased by a subsequent detection reaction using Salmonella-specific oligonucleotides as probes. It is also possible to use non-specific oligonucleotides as probes.
• amplification can also be carried out in the presence of one or more non-specific oligonucleotides, so that possibly also DNA/RNA of other microorganisms that are not to be detected may be amplified.
• Such an amplification process is generally less specific and should therefore be backed up by a subsequent detection reaction using Salmonella-specific oligonucleotides as probe.
• amplification products formed in the indirect processes are known to the person skilled in the art. These include, inter alia, visualisation by means of gel electrophoresis, the hybridisation of probes on immobilised reaction products [coupled to nylon or nitrocellulose filters (“Southern blots”) or, for example, on beads or microtitre plates] and the hybridisation of the reaction products on immobilised probes (e.g. “reverse dot blots” or beads or microtitre plates coupled with probes).
• Salmonella-specific or non-specific oligonucleotides for use as probes and/or primers in direct or indirect detection processes can be labelled or modified. They may comprise, for example, radioactive, coloured or fluorescent groups or groups that enable immobilisation on a solid phase or groups that have been modified or that modify in some other way, such as, for example, antibodies, antigens, enzymes or other substances having an affinity for enzymes or enzyme complexes. Probes and primers may be either naturally occurring or synthetically produced double- or single-stranded DNA or RNA or modified forms of DNA or RNA, such as, for example, PNA (in those molecules the sugar units have been replaced by amino acids or peptides).
• PNA in those molecules the sugar units have been replaced by amino acids or peptides
• nucleotides or a number of nucleotides of the probes or primers may be replaced by analogous building blocks (such as, for example, nucleotides that do not naturally occur in the target nucleic acid).
• the detection can be carried out also by means of an internally labelled amplification product. That can be effected, for example, by the integration of modified nucleoside triphosphates (e.g. coupled with digoxygenin or fluorescein) during the amplification reaction.
• Suitable Salmonella-specific oligonucleotides according to the invention are nucleic acids, preferably from 15 to 30 bases long, that correspond, at least in a 10 base long sequence, to sequences 1 to 10 or to their complementary sequences. Relatively small differences (1 or 2 bases) in that 10 base long sequence are possible without loss of the requisite specificity in the amplification and/or hybridisation. The person skilled in the art will know that in the case of such relatively small differences the reaction conditions need to be altered accordingly.
• DNA of the selected Salmonella strains was prepared by standard procedures and the relevant region was amplified by PCR and subsequently sequenced. In the PCR and the subsequent sequencing, the following primers were used for most of the Salmonella strains:
• degenerate primers or primers having deoxyinosin at the variable sites there are customarily used degenerate primers or primers having deoxyinosin at the variable sites.
• a number of degenerate oligonucleotides that were potentially suitable as primers for the detection of all Salmonella enterica subspecies were therefore deduced from the above-mentioned sequence comparison. It was found, however, that those degenerate primers have only limited suitability for PCR detection since they result in an increase in the occurrence of non-specific reaction products, especially in the case of sequence regions of high complexity. Since the sensitivity of the PCR detection generally suffers from the occurrence of such non-specific reaction products, a different procedure was tried. “Complementing” primers were used in the PCR.
• the advantage over degenerate primers lies in the lesser complexity of the primer mixture according to the invention, as a result of which the probability that non-specific amplification products will be formed is markedly reduced. As has been shown in a number of experiments, this is especially advantageous in PCR detection using samples having a high content of “non-specific” DNA (DNA that does not come from bacteria to be detected) since, otherwise, the sensitivity of the detection may be radically reduced.
• a major advantage when using complementing oligonucleotides/primers lies in the possibility of optimising existing detection processes. For example, it is possible that individual false negative results can be eliminated by additionally using in the PCR and/or hybridisation reaction oligonucleotides comprising the sequence of the previously undetected strains.
• the DNA sequence comparison yielded a number of relatively short DNA regions that appeared to be potentially suitable for the strategy described (use of in total ⁇ 3 primers in the PCR) for optimising the Salmonella detection process.
• the following Example is given by way of clarification.
• Section I (position 1336 to 1355 of SEQ ID NO: 1 in WO 95/00664)
• Section II position 1342 to 1361 of SEQ ID NO: 1 in WO 95/00664.
• CAGAATACGCCCCGTTCGGC SEQ ID NO:3 CAGAATACACCCCGTTCGGC SEQ ID NO:4
• Section III (complementary to position 1483 to 1502 of SEQ ID NO: 1 in WO 95/00664)
• CAACCTAACTTCTGCGCCAG SEQ ID NO:6 CAACCTAACTTCTGCACCAG SEQ ID NO:7 CAACCTAACCTCTGCGCCAG SEQ ID NO:8 CAACCTAACTTCTGCGGCAG SEQ ID NO:9 CAACCTAACTTCTGCGGCAG SEQ ID NO:10
• oligonucleotides Sa 1 to 10 were used in the PCR in the following combinations:
• Primer combination 1 Sa1/Sa2 (each in a final concentration of 0.2 ⁇ M) Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08 ⁇ M)
• Primer combination 2 Sa3/Sa4/Sa5 (each in a final concentration of 0.13 ⁇ M) Sa6/Sa7/Sa8/Sa9/Sa10 (each in a final concentration of 0.08 ⁇ M)
• DNA was isolated by standard processes from pure cultures of the Salmonella strains listed in Table 1a. Approximately from 10 to 100 ng of each of those DNA preparations was then used in the PCR in the presence of primer combination 1 or primer combination 2, 200 ⁇ M of dNTP's (Boehringer Mannheim), 1.5 mM MgCl 2 , 16 mM (NH 4 ) 2 SO 4 , 67 mM Tris/HCl (pH 8.8), 0.01% Tween 20 and 0.03 U/ ⁇ l Taq-polymerase (Biomaster). The PCR was carried out in a Perkin-Elmer 9600 thermocycler having the following thermoprofile:
• the amplification products were separated by means of agarose gel electrophoresis and visualised by staining with ethidium bromide.
• the expected product of 167 bp length (primer combination 1) or of 161 bp length (primer combination 2 was observed in all cases in which DNA of strains of the Salmonella genus was present (compare Table 1a), but not in the presence of DNA of other tested bacteria (compare Table 1b).
• the DNA contained in the gels was transferred by standard methods to nylon filters and hybridised with the oligonucleotide ST14 (TTTGCGACTATCAGGTTACCGTGG (SEQ ID NO:13) (see claim 3, WO 95/00664)) labelled at the 5′ end with digoxygenin to test the base specificity especially sensitively.
• Hybridisation was effected in 5 ⁇ SSC, 2% blocking reagent, 0.1% lauryl sarcosine, 0.02% SDS and 5 pmol/ml of probe for 4 hours at 60° C. Washing was carried out in 2 ⁇ SSC, 0.1% SDS for 2 ⁇ 15 minutes at 60° C.
• Detection was carried out according to standard methods using anti-digoxygenin/alkaline phosphate conjugates in the presence of 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro-blue tetrazolium chloride (Boehringer Mannheim).
• IIIb 16 enterica D 1 IIIb 1, 9, 12: y: z 39 subsp.
• I IIIb 16 k: ⁇ diarizonae J IIIb 17: z 10 , e, n, x, z 15 O IIIb 35: k: e, n, z 15 P IIIb 38: 1, v: z 53 IIIb 38: 1, v: z 54 T IIIb 42: k: z 35 X IIIb 47: b: z 6 IIIb 47: k: z 35 IIIb 47: r: z 53 IIIb 47: ⁇ : ⁇ Y IIIb 48: (k): z 53 Z IIIb 50: k: z IIIb 50: r: z O: 53 IIIb 53: 1, k: z O: 60 IIIb 60: z 52 : z 53 O: 61 IIIb 61: 1: z IIIb 61: 1, v: 1, 5, 7 IIIb 61: 1, v: 1, 5, 7: (z
• ATCC 20182 Listeria monocytogenes ATCC 19118 Pediococcus domnatus IfGB 0101 Proteus vulgaris DSM 2041 Pseudomonsas fluorescens DSM 6290 Serratia marcescens IfGB 0101 Shigella flexneri DSM 4782 Staphylococcus aureus ATCC 6538 Yersinia enterocolitica DSM 4780

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